The long-term objective of this proposal is to develop a reliable microscopic assay for evaluating the viability and developmental capacity of mammalian oocytes used for in vitro fertilization and embryo transfer. The specific aims are: (1) the selection and use of vital fluorescent stains to study changes in cellular structure that occur during oocyte maturation, in vitro fertilization, and early embryogenes in rodents that may provide an accurate, non-toxic, in vitro assay for developmental potential; this approach relies heavily on the use of time lapse video image intensification microscopy for the enhancement and detection of vital stain fluorescence and may be generally applicable for the assessment of in vitro fertilization and embryo transfer in domestic animals and humans; (2) the timing and topography of morphological events during rodent oocyte maturation and fertilization in vitro will be determined from time lapse video recordings of living material; it is hoped that specific patterns in organelle (nuclei, lysosomes, mitochondria) distribution or staining properties might be used to establish the precise sequence and character of cellular events required for normal development; this information may be useful in evaluating oocyte maturity, polyploidy or other developmental abnormalities; (3) the role of the cytoskeleton during the meiotic maturation of rat, mouse, and pig oocytes in culture will be evaluated with fluorescence microscopy and pharmacological agents; multiple fluorochrome labeling experiments will define the spatial and temporal associations of microtubules and actin microfilaments during nuclear maturation and specific cytoskeleton-modifying drugs will be used to analyze their effects on living oocytes undergoing maturation; these results may suggest new avenues of research for the experimental manipulation of domestic animal oocytes as well as provide basic information on the cellular regulation of meiotic maturation.